Acylation of pyrazofurin

ABSTRACT

A process for preparing new compounds which are N- and O-acylates of pyrazofurin comprises first selective mono-N-acylation under non-basic conditions in an organic solvent. The mono-N-acylate so formed is further acylated under mild basic conditions to provide a tetra-acylated or penta-acylated pyrazofurin derivative, depending upon the duration of reaction. Mild solvolysis of either a tetra-acylate or a penta-acylate provides a tri-acylated pyrazofurin derivative. In the presence of a strong base, the mono-N-acylate is further acylated to provide different tetra-acylates or penta-acylates of pyrazofurin, again depending upon the duration of reaction. Pyrazofurin acylates are useful as antiviral, antipsoriatic, and antifungal agents, as well as intermediates for new C-nucleosides.

This is a division of application Ser. No. 490,627, filed July 22, 1974,now U.S. Pat. No. 3,960,836.

BACKGROUND OF THE INVENTION

Pyrazofurin is a C-nucleoside antibiotic,4-hydroxy-3-β-D-ribofuranosylpyrazole-5-carboxamide, obtained initiallyby fermentation of a strain of Streptomyces candidus. In accordance withthe nomenclature of the United States Adopted Names Council, pyrazofurinis the generic name which replaces the former generic name pyrazomycin.Methods for the production, recovery, purification, and characterizationof pyrazofurin are described in detail in U.S. Pat. Nos. 3,674,774 and3,802,999. Pyrazofurin has been shown to possess antiviral activity, forexample against rhinovirus, measles, herpes simplex, and vacciniaviruses. In addition, pyrazofurin has exhibited antitumor activityagainst several carcinomas, as demonstrated by Sweeney, el al., CancerResearch, 33, 2619-2623 (1973).

Extensive research has been directed to the study of antiviral agentsand to potential antitumor agents in general. Difficulty has generallybeen encountered in the development of antiviral agents because virusesare intracellular parasites which rely on the metabolic processes of theinvaded call for their own existence. Consequently, agents which inhibitor kill the viruses may in addition injure the host cells that harborthem. Similarly, the severe systemic toxicity of many potentially usefulantitumor agents generally limits their use.

Azauridine is one such antitumor agent which has been used successfullyto produce partial remissions of acute leukemias in adults; however, theresults obtained are generally only temporary. Large intravenous dosesare required because of its poor absorption from the intestine, andsustained blood levels are difficult to maintain due to its rapidexcretion. An orally effective triacetyl derivative of azauridine,Azaribine®, has been prepared in an effort to increase effective bloodlevels of free azauridine. However, moderate anemia and neurologicaldisturbances are common side effects accompanying triacetylazauridineusage.

Considerable interest has recently been directed to pyrazofurin, notonly because of its antiviral and antitumor activities, but also becauseit is one of very few C-nucleosides showing antitumor activity. Thecomplete chemical synthesis of pyrazofurin has recently been reported byFarkas Flegelova, and Sorm, Tetrahedron Letters, 2279-2280 (1972).Panzica and Townsend have prepared several nucleosides which arestructurally similar to pyrazofurin and which also display antitumoractivity, Journal of Organic Chemistry, 36, 1594-1596 (1971).

It is an object of the present invention to provide novel C-nucleosideswhich are useful pharmacological agents. In particular, it is an objectof this invention to provide certain acylated derivatives ofpyrazofurin. Additionally, because normal acylation conditions whenapplied to pyrazofurin lead to very complex mixtures of partial andtotal acylates, it is a further object of this invention to provideprocesses whereby various partial acylates of pyrazofurin are cleanlyprepared. A still further object of this invention is to provide newcompounds which display antiviral, antifungal, and antitumor activity,and are additionally useful as antipsoriatic agents.

SUMMARY OF THE INVENTION

This invention relates to certain acylated derivatives of pyrazofurinand to processes for their preparation. The invention is particularlydirected to the preparation of certain O- and N-acylated pyrazofurinderivatives. More particularly, the invention is directed to certainmono-, di-, tri-, tetra-, and penta-acylates of pyrazofurin. Thecompounds provided by this invention have the formula ##STR1## in whichR₁, R₂, R₃, and R₅ independently are hydrogen or C₁ -C₆ alkanoyl and R₄is hydrogen, C₁ -C₆ alkanoyl, palmitoyl, benzoyl, or adamantoyl, withthe limitations that at least one of R₁, R₂, R₃, R₄, or R₅ is a groupother than hydrogen, that R₂ is alkanoyl only when R₁ is alkanoyl, thatR₃ is alkanoyl only when R₄ is alkanoyl; and that R₅ is alkanoyl onlywhen R₃ and R₄ are both alkanoyl.

The compounds of this invention are prepared by the novel process whichcomprises as a first step the selective monoacylation of pyrazofurinwith an acylating agent under non-basic conditions in an organic solventto provide the mono-N₁ -acylate. Further acylation of the mono-N₁-acylate under mild basic conditions for moderate periods of timeprovides a tetra-acylate, specifically a 2',3',5' -tri-O-acylate-N.sub.1 -acylate. When the mono-N₁ -acylate is acylated under mild basicconditions for prolonged periods of time, a penta-acylate is recovered,namely a 4,2',3',5'-tetra-O-acylate-N.sub. 1 -acylate. Solvolysis ofeither the tetra-acylate or the penta-acylate affords a 2',3',5'-tri-O-acylate. Acylation of the mono-N₁ -acylate under more vigorousacylation conditions in the presence of a stronger base provides adifferent tetra-acylate, specifically one wherein the 5-carboxamidegroup is acylated along with the three hydroxyl groups of theribofuranosyl moiety. By extending the length of reaction under the morebasic acylation conditions, a different penta-acylated pyrazofurinderivative is formed, namely a 2',3',5'-tri-O-acylate-N.sub. 1 -acylatewherein the 5-carboxamide group is also acylated.

DETAILED DESCRIPTION OF THE INVENTION

As hereinbefore indicated, the compounds of this invention have theformula ##STR2## R₁ in the above formula is hydrogen or C₁ -C₆ alkanoyl.Examples of C₁ -C₆ alkanoyl groups include both straight and branchedchain carboxylic acid residues having no more than six carbons, such asformyl, acetyl, propionyl, n-butyryl, isobutyryl, n-pentanoyl,2-methylbutyryl, pivaloyl, n-hexanoyl, 3-methylpentanoyl,2,3-dimethylbutyryl, and the like. The most preferred alkanoyl groupsare the straight chain alkanoyl groups such as acetyl or butyryl, forexample.

R₂ in the above formula is hydrogen or C₁ -C₆ alkanoyl, but is alkanoylonly when R₁ is alkanoyl. When R₂ is alkanoyl, it is preferably the sameas R₁.

R₃ in the above formula is hydrogen or C₁ -C₆ alkanoyl, but is alkanoylonly when R₄ is alkanoyl. Both R₃ 's together can be a protecting groupfor the 2' and 3' hydroxyl groups of the ribofuranosyl ring system."Protecting group" as used herein refers to common glycol protectinggroups such as cyclic acetals and ketals, cyclic esters and orthoesters.Examples of typical protecting groups commonly used includeisopropylidene ketal, benzylidene acetals, cyclohexylidene ketal, cycliccarbonates, thiocarbonates, and the like.

R₄ in the above formula is hydrogen, C₁ -C₆ alkanoyl, palmitoyl,benzoyl, or adamantoyl.

R₅ in the above formula is hydrogen, or when R₃ and R₄ are alkanoyl, R₅can be C₁ -C₆ alkanoyl.

At least one of R₁, R₂, R₃, R₄, or R₅ of the above formula is other thanhydrogen.

The compounds of the present invention are named according to standardnaming systems by following the numbering system shown in the aboveformula. Pyrazofurin is described by the above formula when R₁, R₂, R₃,R₄, and R₅ are all hydrogen, and is systematically named4-hydroxy-3-β-D-ribofuranosylpyrazole-5-carboxamide. As can be seen fromthe above formula, pyrazofurin is a C-nucleoside which contains sixcenters which are capable of reacting with acylating agents. Inparticular, the pyrazole ring portion of pyrazofurin contains a nitrogenatom which can be acylated. Additionally, the pyrazole ring bears a4-hydroxyl substituent and a 5-carboxamide substituent, both of whichcan be acylated. The ribofuranosyl portion of pyrazofurin can formacylates at each of its three hydroxyl groups. It should be recognizedthat any reactive nitrogen or hydroxyl group of pyrazofurin can beacylated with essentially any acylating agent under appropriateconditions. For example, any acyl group can be attached at thepyrazole-N₁ -position if desired.

The compounds of the present invention are prepared by selectiveacylation of pyrazofurin under controlled conditions of basicity,reaction temperature, and duration of reaction. Depending upon theconditions of acylation, certain pyrazofurin mono-acylates, di-acylates,tri-acylates, tetra-acylates, and penta-acylates are prepared andisolated in good yield.

The normal procedure for preparing acylated derivatives ofpolyfunctional compounds such as pyrazofurin generally comprisesexhaustive acylation in the presence of an acid binding agent. Forexample, lincomycin is fully acylated in the presence of an acylatingagent and a base, as described in U.S. Pat. No. 3,318,866. When thisgeneral procedure is applied to pyrazofurin, a complex mixture ofacylates comprising the mono-, di-, tri-, tetra-, penta-, andhexaacylates are obtained in admixture with each other. Separation andpurification of the individual partial acylates is extremely difficultand inefficient. Moreover, the yields of any particular partial acylateare quite low due to the concomitant production of other partialacylates as well as completely acylated products. Additionally, basicsolutions of pyrazofurin are known to equilibrate between differenttautomeric forms of pyrazofurin, thus further complicating normalacylation.

In accordance with the invention, pyrazofurin is selectivelymono-acylated at the pyrazole-N₁ -position, thereby freezing thestructure into one tautomeric form. Once the mono-N₁ -acylate has beenformed in accordance with the present invention, subsequent specificacylations are greatly simplified. In particular, certain di-, tri-,tetra-, and penta-acylates are conveniently prepared from the mono-N₁-acylate.

in one aspect of the present invention, pyrazofurin is first treatedwith an acylating agent in an organic solvent, in the absence of a base,to provide the corresponding mono-N₁ -acylate. Any of a number ofacylating agents can be used in the present process, the nature of theparticular agent selected not being critical to the process. Typicalacylating agents commonly used include acid halides, especially acidchlorides and acid bromides and most especially acid chlorides; acidanhydrides, including mixed acid anhydrides; ketenes, and the like.Generally, the acid anhydrides are the preferred acylating agents whenthe lower alkanoyl groups, such as acetyl, propionyl, and butyryl forexample, are desired. When larger acyl groups such as benzoyl,palmitoyl, or adamantoyl for example, are desired, the correspondingacid halide is generally preferred. Examples of acid anhydridesroutinely used include acetic anhydride, formic acetic anhydride,propionic anhydride, butyric anhydride, hexanoic anhydride, and thelike. Typical acid halides which can be used include acetyl chloride,butyryl bromide, 3-methylpentanoyl chloride, benzoyl chloride,adamantoyl chloride, palmitoyl chloride, and the like. Examples ofuseful ketenes include ketene, ethylketene, propylketene, butylketene,isopropylketene, isobutylketene, and the like.

The mono-N₁ -acylation reaction is carried out in an organic solventsuch as an alcohol, for example. An alcohol such as methanol or ethanolis especially preferred when acylation of the hydroxyl groups of theribofuranosyl portion of purazofurin is to be avoided, as in the case ofmono-N₁ -acylation for example. The alcoholic solvents are generallymore reactive toward acylation than are the ribofuranosyl hydroxylgroups, and consequently will react with any excess acylating agent inthe reaction mixture more rapidly than will the ribofuranosyl hydroxylgroups. Other solvents that can be used, for example as co-solvents ifdesired, include ketones such as acetone or methyl ethyl ketone; etherssuch as diethyl ether or tetrahydrofuran, and the like. The temperatureof the mono-N₁ -acylation reaction is generally maintained below about40° C. The temperature is most conveniently maintained between about 0°to about 30° C., and in practice, the temperature is preferablymaintained at about 0° to 25° C. The reactants, that is pyrazofurin andthe desired acylating agent, can be commingled in about equimolaramounts; however, excess acylating agent is generally preferred. Theacylating agent, preferably an acid anhydride or a ketene, is generallyemployed in about a 2 to 20 molar excess; however, more can be used ifdesired. Alternatively, when an acid halide is selected as the acylatingagent, an approximately equimolar amount is preferably used. The mono-N₁-acylation reaction is substantially complete after about 30 to about 90minutes, especially when carried out at the preferred temperature ofabout 0° to 25° C. The pyrazofurin mono-N₁ -acylate is convenientlyisolated by removal of any solvent and excess acylating agent present inthe reaction mixture, generally by evaporation. The product can befurther purified, if desired, by standard procedures such aschromatography, crystallization, distillation, or the like. It should benoted, however, that the mono-N₁ -acyl group is in general easilysolvolyzed with protic solvents, especially water, and should thereforenot be exposed to such conditions.

Illustrative examples of mono-N₁ -acylates of pyrazofurin provided bythe present invention include:

4-Hydroxy-3-β-D-ribofuranosyl-N₁ -acetylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-ribofuranosyl-N₁ -n-butyrylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-ribofuranosyl-N₁ -n-hexanoylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-ribofuranosyl-N₁-(2-methylbutyryl)-pyrazole-5-carboxamide; and the like.

The mono-N₁ -acylates of pyrazofurin are useful as antiviral agents,antitumor agents, and antipsoriatic agents. Additionally, the mono-N₁-acylates are useful as intermediates for the preparation of otheracylated pyrazofurin derivatives, as described hereinbelow.

In another aspect of this invention, a pyrazofurin mono-N₁ -acylate isfurther acylated in the presence of a weak base, thereby providingeither a tetra-acylate or a penta-acylate, depending upon the durationof the reaction. It should be understood that when more than one acylgroup is present in a pyrazofurin derivative, it is possible to formmixed acylates, that is to say, acylates wherein not all of the acylgroups are the same.

The pyrazofurin mono-N₁ -acylate is further acylated in the presence ofa weak base. The term "weak base" refers to a base having a pK'_(b) inthe range of about 7 to about 10. Typical weak bases commonly usedinclude amines, especially arylamines and cyclic amines such aspyridine, dimethylaniline, piperazine, p-toluidine, and the like.Pyridine is an especially preferred weak base in the present process.The amount of base used is not critical, however, an excess of base isgenerally used. The amount of base generally is sufficient to serve assolvent or co-solvent. Alternatively, the acylation can be carried outin any of a number of organic solvents, if desired. If a solvent isdesired, one can be selected from among the aromatics, such as benzeneor toluene for example; or the halogenated hydrocarbons such asmethylene chloride or chloroform; or the ethers, such as diethyl ether,tetrahydrofuran, or the like. These solvents can serve as co-solvents ifdesired, for example in conjuncton with the acylating agent and thebase. The acylation is most conveniently carried out by using simply theweak base and the acylating agent as the reaction medium. Morespecifically, the acylation can best be carried out by commingling theacylating agent and the mono-N₁ -acylate in an excess of a weak base,preferably pyridine, and stirring the reaction mixture for a timesufficient to effect either tetra-acylation or penta-acylation. Theacylating agent, preferably an acid anhydride as described hereinabove,is used in excess of the mono-N₁ -acylate. The excess normally amountsto about 10 to 10 molar excess; however, more can be used if desired.The preparation of the pyrazofurin tetra-acylated derivative issubstantially complete after about 1 to about 3 hours when the reactionis carried out at a temperature of about -10° to about 15° C. Thetemperature can be increased if desired, for example, to about 25° C.;however, the reaction can best be controlled if the temperature is keptbelow about 15° C., preferably at about 0° C. The tetra-acylate that isformed is a pyrazofurin 2', 3', 5' -tri-O-acylate-N₁ -acylate.

The preferred tetra-acylates are those wherein all four acyl groups areall the same alkanoyl group. The tetra-acylate is generally isolated bycomplete removal of any excess base, excess acylating agent, or anysolvents present in the reaction mixture. Complete removal of any excessbase or excess acylating agent can best be accomplished by repeatedlydissolving the product mixture in a suitable organic solvent or solventmixture and subsequently distilling the solvent, thereby effectivelyremoving any excess base or acylating agent. Solvents generally used forthis purpose are aromatic solvents, such as benzene, toluene, or xylene,or alcohols such as methanol or ethanol. Mixtures of solvents can beused if desired; for example, a mixture of methanol and benzene orbenzene and toluene is desirable. Once all of the excess acylating agentand excess base and any other solvent has been completely removed fromthe tetra-acylate, further purification of the product is generally notneeded. Further purification can be accomplished if desired, however, bystandard methods such as gas chromatography, column chromatography, andthe like.

Typical examples of pyrazofurin 2', 3', 5' -tri-O-acylate-N₁ -acylatesprepared by the above-described process include, among others:

4-Hydroxy-3-β-D-(2',3',5' -tri-O-acetylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3',5' -tri-O-n-butyrylribofuranosyl)-N₁-n-butyrylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3',5' -tri-O-n-hexanoylribofuranosyl)-N₁-n-hexanoylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3',5' -tri-O-isobutyrylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3',5' -tri-O-n-butyrylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3',5' -tri-O-isobutyrylribofuranosyl)-N₁-isobutyrylpyrazole-5-carboxamide; and the like.

Mixed tetra-acylates, wherein R₁, R₃, and R₄ in the above generalformula are different acyl groups, are prepared by mono-N₁ -acylation ofa protected pyrazofurin derivative, such as a 2',3'-acetonide ofpyrazofurin for example. The protected mono-N₁ -acylate is then acylatedat the 5'-position to provide the corresponding protected di-acylate,which is subsequently converted to the pyrazofurin mono-5'-0-acylate.The 5'-mono-0-acylate is then N₁ -acylated and then further acylated atthe 2' and 3' positions to provide the desired tetra-acylate. Morespecifically, pyrazofurin is converted to a 2',3'-protected derivativewith any of a number of suitable protecting groups. A preferredprotecting group, for example, is the isopropylidine group. Generally,pyrzaofurin can simply be treated with acetone in the presence of anacid, such as p-toluenesulfonic acid for instance, to provide thedesired pyrazofurin 2',3'-acetonide. The protected pyrazofurin is thenacylated at the N₁ -position of the pyrazole ring, for example with anacylating agent such as an acid anhydride, under non-basic conditionsand in a protic solvent such as methanol. The pyrazofurin mono-N₁-acylate 2',3'-acetonide so formed is subsequently further acylated atthe 5'-position, for example with an acylating agent such as an acidanhydride or acid halide, in the presence of a weak base such aspyridine, for instance, thus providing a protected diacylatedpyrazofurin derivative. Both the N₁ -acyl group and the2',3'-isopropylidine protecting groups are readily removed by solvolysisin a protic solvent, such as methanol or water for example. Generally,the 2',3'-isopropylidine group and the N₁ -acyl group are solvolyzedwhen the pyrazofurin di-acylated acetonide is stirred in a proticsolvent at about 30 to about 80° C. for a period of time of about 2 toabout 20 hours. Removal of the solvent provides the product, a4-hydroxy-3-β-D-(5'-0-acylribofuranosyl)pyrazole-5-carboxamide.

Examples of mono-5'-0-acylates of pyrazofurin include:

4-Hydroxy-3-β-D-(5'-0-acetylribofuranosyl)pyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(5' -O-benzoylribofuranosyl)pyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(5' -O-adamantoylribofuranosyl)pyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(5' -O-palmitoylribofuranosyl)pyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(5' -O-butyrylribofuranosyl)pyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(5' -O-n-hexanoylribofuranosyl)pyrazole-5-carboxamide;and the like.

Acylation of the pyrazofurin mono-5'-0-acylate under non-basicconditions and in a protic solvent, such as methanol for example,provides a pyrazofurin di-acylated derivative, namely a4-hydroxy-3-β-D-(5' -O-acylribofuranosyl)-N₁-acylpyrazole-5-carboxamide. Further acylation of the di-acylate, forexample with an acid anhydride in the presence of a weak base such aspyridine, provides the corresponding tetra-acylated pyrazofurinderivative, namely a 2',3',5' -tri-O-acylate-N₁ -acylate. By selectingdifferent acylating agents at each step, a mixed tetra-acylate can beprepared if desired. Examples of mixed tetra-acylates include:

4-Hydroxy-3-β-D-(2',3' -di-O-acetyl-5'-0-n-butyrylribofuranosyl)-N₁-propionylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3' -di-O-acetyl-5'-0-palmitoylribofuranosyl)-N₁-n-butyrylpyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3' -di-O-n-butyryl-5'-0-acetylribofuranosyl)-N₁-pivaloylpyrazole-5-carboxamide; and the like.

In another aspect of the invention, the mono-N₁ -acylated derivative ofpyrazofurin is further acylated to provide a penta-acylate, specificallya 4,2',3',5' -tetra-O-acylate-N₁ -acylate of pyrazofurin. The reactiongenerally is carried out under conditions similar to those used in thepreparation of the tetra-acylate. In particular, a weak base such aspyridine is used when reacting the mono-N₁ -acylate with thecorresponding acylating agent, a preferred anhydride for instance,either in a solvent or by using the base and the acylating agent as thesolvent. The reaction normally is conducted over a longer period oftime, about 5 to about 20 hours, and the temperature generally ismaintained at about 20° to about 30° C. Longer reaction times can beincorporated if desired. The acylating agent is incorporated in excessof the mono-N₁ -acylate pyrazofurin derivative, generally in about a 10to 20 molar excess; however, even more can be used if desired.Similarly, the base is used in excess, preferably in amounts sufficientto serve as a co-solvent with the acylating agent. The productpenta-acylate is isolated by complete removal of any excess acylatingagent, base, solvent, or the like, and usually the product needs nofurther purification. If desired, however, the pyrazofurin penta-acylatecan be washed with aqueous solutions of acid or base, such as dilutemineral acid or dilute bicarbonate bases, for example. The product canbe further purified if desired by chromatography and the like.

As hereinbefore indicated, the preferred penta-acylates are thosewherein all acyl substituents are the same. Typical examples of suchpreferred penta-acylates include:

4-Acetoxy-3-β-D-(2',3',5' -tri-O-acetylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide,

4-n-Butyryloxy-3-β-D-(2',3',5' -tri-O-n-butyrylribofuranosyl)-N₁-n-butyrylpyrazole-5-carboxamide;

4-Isohexanoyloxy-3-β-D-(2',3',5' -tri-O-isohexanoylribofuranosyl)-N₁-isohexanoylpyrazole-5-carboxamide; and the like.

Mixed penta-acylates can be prepared by selecting the appropriatestarting materials. For example, acylation of a mono-N₁ -acylate couldprovide a penta-acylate of the above formula wherein R₂, R₃, and R₄ areall the same acyl group, which is different from R₁. Further acylationof a mixed tetra-acylate, wherein R₁ is different from R₃ and R₄,provides a mixed penta-acylate wherein R₁ is different from R₃ and R₄,which are different from R₂. Examples of mixed penta-acylates include:

4-Acetoxy-3-β-D-(2',3',5'-tri-O-n-butyrylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide;

4-Isopentanoyloxy-3-β-D-(2',3'-di-O-acetyl-5'-adamantoylribofuranosyl)-N₁-n-butyrylpyrazole-5-carboxamide; and

4-n-Hexanoyl-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)-N₁-n-butyrylpyrazole-5-carboxamide.

As indicated hereinabove, the N₁ -acyl group and the 4-acyl group of apyrazofurin polyacylate generally are somewhat labile to protic solventsand are susceptible to cleavage by solvolysis. Consequently, either thetetra-acylates or the penta-acylates, which are prepared as describedhereinbefore, can be converted to tri-O-acylates by simple solvolysis ina suitable protic solvent. Typical solvents useful for such solvolysisreactions include water, alcohols, such as methanol or ethanol forinstance, or mixtures of such protic solvents. The reaction generally iscarried out by stirring the appropriate pyrazofurin tetra-acylate orpenta-acylate in a protic solvent, preferably methanol or a methanol andwater mixture, at an elevated temperature for several days. Moreparticularly, the temperature of the reaction mixture is normallymaintained below about 120° C., preferably in the range of about 30° toabout 100° C. The reaction is conveniently carried out at the refluxtemperature of the appropriate solvent. The solvolysis is normallysubstantially complete after about 1 to 10 days, depending to someextent upon the particular acyl group being cleaved. The reactiongenerally is allowed to continue for about 4 or 5 days. The product, apyrazofurin 2',3',5'-tri-O-acylate, is recovered by complete removal ofany reaction solvent. Further purification is generally not needed;however, further purification by procedures such as solid-liquidchromatography, thick-layer chromatography, crystallization, or thelike, can be carried out if desired. While mixed tri-O-acylates can beprepared from the corresponding mixed tetra- or penta-acylate, thepreferred tri-O-acylates are those wherein all three acyl groups are thesame C₁ -C₆ alkanoyl group. Examples of preferred tri-O-acylates ofpyrazofurin include:

4-Hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)-pyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3',5'-tri-O-n-butyrylribofuranosyl)pyrazole-5-carboxamide;

4-Hydroxy-3-β-D-(2',3',5'-tri-O-isohexanoylribofuranosyl)pyrazole-5-carboxamide.

Further in accordance with the present invention, a pyrazofurin mono-N₁-acylate can be acylated under the more vigorous acylation conditions ofa strong base to provide tetraacylates and penta-acylates wherein the5-carboxamide group is acylated. A "strong base" as used herein refersto a base having a pK'_(b) in the range of about 3 to about 4. Typicalstrong bases for the present process include certain amines such astriethylamine, isopropylamine, methyldiethylamine, ethylamine,dimethylamine, isoamylamine, and the like. Triethylamine is anespecially preferred strong base in the present process. The amount ofbase used in the reaction is not critical; however, the base is usuallyused in excess of the mono-N₁ -acylate starting material, and generallyin amounts sufficient to serve as solvent or co-solvent, in conjunctionwith the acylating agent. An additional solvent or co-solvent can beincorporated if desired. Suitable solvents include organic solvents suchas aromatics, ethers, esters, amides, halogenated hydrocarbons, and thelike. Typical solvents include, for example, benzene, toluene,dimethylacetamide, diethyl ether, ethyl acetate, chloroform, and thelike.

Generally, the pyrazofurin mono-N₁ -acylate and the acylating agent arecommingled in a suitable strong base, such as triethylamine for example.The acylating agent is normally employed in amounts in excess of themono-N₁ -acylate, usually in about 10 to 20 molar excess. More acylatingagent, or less, can be used if desired; however, at least a 4 molarexcess is generally needed. The acylating agent, especially an acidanhydride, and the base are generally used in excessive amounts such asto serve as reaction solvent. When the acylating agent selected is anacid halide, the excess amounts of acylating agent is usually kept to aminimum, for example about a 4 molar excess. By varying the length ofreaction, either a pyrazofurin tetra-acylate or a pyrazofurinpentaacylate is obtained, either of which is acylated at thepyrazole-5-carboxamide group. More specifically, when the acylationreaction is carried out at a temperature of about 0° to about 15° C.,the pyrazofurin 2',3',5'-tri-O-acylate wherein the 5-carboxamide groupis also acylated is obtained after a period of time of about 1/2 toabout 2 hours. The temperature can be increased if desired, for exampleto about 25° C., but the reaction is best carried out at the lowertemperature of about 0° to 15° C. The product is isolated by completeremoval of any excess acylating agent, base, or other solvents.Generally, the product so obtained can be stirred in a protic solvent,such as aqueous methanol for example, thereby insuring completeconversion of any penta-acylate to the corresponding tetra-acylate.Further purification by standard procedures such as solid-liquidchromatography or thick layer chromatography can be carried out ifdesired. The preferred tetra-acylates are those wherein all four acylgroups are the same and are selected from among C₁ -C₆ alkanoyl. Typicalexamples of preferred tetra-acylates of pyrazofurin include:

4-Hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)-pyrazole-5-N-acetylcarboxamide;

4-Hydroxy-3-β-D-(2',3',5'-tri-O-n-butyrylribofuranosyl)pyrazole-5-N-n-butyrylcarboxamide;

4-Hydroxy-3-β-D-(2',3',5'-tri-O-isohexanoylribofuranosyl)pyrazole-5-N-isohexanoylcarboxamide;and the like.

Mixed tetra-acylates can be prepared if desired by selecting the properacylating agent and further acylating a pyrazofurin2',3',5'-tri-O-acylate-N₁ -acylate in the presence of a strong base suchas triethylamine.

In yet another aspect of the invention, a mono-N₁ -acylate ofpyrazofurin can be acylated under conditions of strong base attemperatures of about 20° to 30° C. for longer periods of time, toprovide a penta-acylate, namely, a pyrazofurin 2',3',5'-tri-O-acylate-N₁-acylate wherein the carboxamide group is also acylated. Morespecifically, a pyrazofurin mono-N₁ -acylate is treated with anacylating agent, for example, an acid anhydride, in the presence of astrong base such as triethylamine, for a period of time from about 20 toabout 100 hours, to provide the corresponding penta-acylate. Theacylating agent is generally used in about 10 to 20 molar excess of thestarting mono-N₁ -acylate, and the base is normally employed inquantities sufficient to serve as solvent, as described hereinbefore.The reaction is normally complete after about 36 hours when conducted ata temperature of about 25° C. Isolation of the penta-acylate isaccomplished by complete removal of the solvent, and furtherpurification is generally not needed, but can be accomplished bystandard procedures such as chromatography if desired. As indicatedhereinabove, the preferred polyacylates of the invention are thosewherein all of the acyl groups present are the same. Typically preferredpenta-acylates of the above formula wherein R₁, R₃, R₄ and R₅ are acylgroups include:

4-Hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)-N₁-acetylpyrazole-5-N-acetylcarboxamide;

4-Hydroxy-3-β-D-(2',3',5'-tri-O-n-butyrylribofuranosyl)-N₁-n-butyrylpyrazole-5-N-n-butyrylcarboxamide;

4-Hydroxy-3-β-D-(2',3',5'-tri-O-isohexanoylribofuranosyl)-N₁-isohexanoylpyrazole-5-N-isohexanoylcarboxamide; and the like.

Mixed penta-acylates can be prepared, if desired, by further acylating amixed tetra-acylate in the presence of a strong base such astriethylamine. As with other mixed acylates, however, these mixedpenta-acylates are sometimes more difficult to purify and, consequently,are the least preferred penta-acylates.

The novel compounds of this invention are active against variousviruses, fungi, and tumors, and are especially useful for the treatmentof psoriasis. The compounds can be formulated so as to facilitateconvenient administration. The compounds are especially suited totopical application when treating psoriasis, due to the substantiallycomplete topical absorption of the compounds of the invention. Theactive ingredient can be employed in combination with one or moreadjuvants, diluents, or carriers. For topical application, the activecomponent is generally commingled with diluents and formulated as anointment or a cream. Typical diluents used in ointment preparationsinclude oleaginous ointment bases such as white petrolatum, polyethyleneglycols, lanolin, and the like. Generally, the compound of the inventionwill be formulated in quantities of about 0.01 to about 2 percent byweight. The compound of the invention is most conveniently formulatedinto tablets or capsules for oral administration. Such tablets orcapsules will consist of a compound of this invention as the activeingredient, generally mixed with a carrier or diluent. Examples ofdiluents or carriers which may be employed in the pharmaceuticalcompositions of the present invention are lactose, dextrose, sucrose,sorbital, mannitol, propylene glycol, liquid paraffin, calciumphosphate, microcrystalline cellulose, gelatin, ethyl lactate, and thelike. A typical tablet or capsule, for example, will contain from about300 to 1000 mg. of active ingredient mixed with a suitable carrier ordiluent. In the treatment of psoriasis, the dosages will be administeredabout once or twice per week. The compounds of the invention can beformulated for parenteral administration, preferably intravenous, with asuitable carrier or diluent. Generally, the active ingredient will beadministered in amounts of about 300 to 1000 mg per patient one or twotimes each week for psoriasis. It should be noted that the amount ofcompound of this invention actually to be administered will bedetermined in light of the revelant circumstances surrounding aparticular case, such as the condition to be treated, the particularcompound selected, the route of administration, and the like.

The preparation of these compounds is more fully described in thefollowing detailed examples. It is to be understood, however, that theexamples are illustrative of the compounds embraced by this inventionand of the methods for their preparation and are not to be construed aslimiting the invention to the particular compounds or methodsspecifically described.

EXAMPLE 1 4-Hydroxy-3-β-D-ribofuranosyl-N₁ -acetylpyrazole-5-carboxamide

A solution of 1.0 g. of 4-hydroxy-3-β-D-ribofuranosylpyrazole-5-carboxamide in 20 cc. of anhydrous methyl alcohol was stirred andcooled to 0° C. To the cold stirred solution was added 5 cc of aceticanhydride dropwise over about 10 minutes. The reaction mixture wasstirred for about 1 hour while the temperature was allowed to increaseto about 25° C. The solvent was removed under reduced pressure toprovide an oily residue which was dissolved in 25 cc. of 50 percentmethanol-toluene. Again the solvent was removed under reduced pressure.This procedure was repeated three times to insure complete removal ofany excess acylating agent. After the final solvents were totallyremoved, 4-hydroxy-3β-D-ribofuranosyl-N.sub. 1-acetylpyrazole-5-carboxamide remained as a white solid.

M/e theory 301; found: 301. nmr (CDCl₃): ##STR3##

EXAMPLE 2

4-Hydroxy-3-β-D-ribofuranosyl-N₁ -n-butyrylpyrazole-5 -carboxamide wasprepared by the procedure of Example 1 from butyric anhydride. nmr (D₂O): (60 Hz, t, 3H, --CH₃)

(100 Hz, m, 2H, --CH₂ --)

(161 Hz, t, 2H, ##STR4##

EXAMPLE 3 4-Hydroxy-3-β-D-(2',3', 5'-tri-O-acetylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide.

A solution of 1.0 g. of 4-hydroxy-3-β-D-ribofuranosyl-N₁-acetylpyrazole-5-carboxamide in 10 cc. of dry pyridine was cooled to 0°C. and stirred while 8 cc. of acetic anhydride was added dropwise overabout 10 minutes. The reaction mixture was stirred for 2 hours at 0° C.,after which time the solvent was removed under reduced pressure toprovide an oily residue. The residue was dissolved in 50 cc. of a 50percent solution of methanol-benzene, and the solvent was removed underreduced pressure. This procedure was repeated three times to insurecomplete removal of excess acylating agent. After complete solventremoval, the residue was dissolved in 25 cc. of water and extractedtherefrom with three 25 cc. portions of chloroform. The chloroformextracts were combined, washed with 25 cc. of 0.1 N hydrochloric acid,25 cc. of aqueous saturated sodium bicarbonate solution, and dried. Thesolvent was removed under reduced pressure to provide 4-hydroxy-3-β-D-(2',3' ,5'-tri-O-acetylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide as a white amorphous solid. ##STR5##

EXAMPLE 4 4-Acetoxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide.

To a cooled solution of 1.0 g. of 4-hydroxy-3-β -D-ribofuranosyl-N₁-acetylpyrazole-5-carboxamide in 10 cc. of dry pyridine was added 8 cc.of acetic anhydride dropwise over about 10-15 minutes. The reactionmixture was allowed to warm to about 25° C. and stirring was continuedfor about 12 hours. The solvent was removed under reduced pressure toprovide an oily residue which was dissolved in 20 cc. of an ice-watermixture. The aqueous solution was extracted three times with 25 cc.portions of chloroform. The chloroform extracts were combined and washedwith 25 cc. of 0.1 N hydrochloric acid solution and the twice with 20cc. portions of aqueous saturated sodium bicarbonate solutions. Afterdrying the organic solution, the solvent was removed under reducedpressure to afford4-acetoxy-3-β-D-(2',3',5'-tri-O-acetyl-ribofuranosyl)-N₁-acetylpyrazole-5-carboxamide as a white solid. ##STR6##

EXAMPLE 5 4-n-Butyroxy-3-β-D-(2',3',5'-tri-O-n-butyrylribofuranosyl)-N₁-n-butyrylpyrazole-5-carboxamide.

A solution of 0.5 g. of 4-hydroxy-3-β-D-ribofuranosyl-N.sub. 1-n-butyrylpyrazole-5-carboxamide in 5 cc. of dry pyridine was stirredand cooled to 0° C. while 4 cc. of butyric anhydride was added dropwiseto the reaction mixture during 5 minutes. The temperature of thereaction mixture was allowed to rise to about 25° C. after the additionof the butyric anhydride was complete, and the reaction mixture wasstirred at 25° C. for 6 hours. The reaction mixture was concentrated todryness under reduced pressure to provide a residue which was dissolvedin 20 cc. of an ice-water mixture and 25 cc. of chloroform. The organiclayer was separated and the aqueous layer was further extracted with two25 cc portions of chloroform. The organic extracts were combined andwashed with 0.1 N hydrochloric acid solution, saturated aqueous sodiumbicarbonate solution, dried, and evaporated to dryness under reducedpressure, providing4-n-butyroxy-3-β-D-(2',3',5'-tri-O-n-butyrylribofuranosyl)-N.sub. 1-n-butyrylpyrazole-5-carboxamide as a colorless oil.

M/e theory: 609; found: 609.

EXAMPLE 6 4-Acetoxy-3-β-D- (2',3',5'-tri-O-acetylribofuranosyl)-N₁-n-butyrylpyrazole-5-carboxamide.

A solution of 329 mg. of 4-hydroxy-3-β-D-ribofuranosyl-N.sub. 1-n-butyrylpyrazole-5-carboxamide in 10 cc. of dry pyridine was stirredand cooled to 0° C. while 5 cc. of acetic anhydride was added dropwiseto the stirred solution during 5 minutes. The reaction mixture waswarmed to 25° C. during 1 hour, and stirred at 25° C. for 4 hours. Thesolvent was removed under reduced pressure, leaving a residue which wasdissolved in 30 cc. of water. The product was extracted into chloroform.The chloroform extracts were combined and washed with saturated aqueoussodium bicarbonate and dried. Removal of the solvent afforded4-acetoxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)-N.sub. 1-n-butyrylpyrazole-5-carboxamide.

EXAMPLE 7 4-Hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)pyrazole-5-carboxamide.

A solution of 5.0 g. of 4-acetoxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)-N₁ -acetylpyrazole-5-carboxamide in 100 cc.of methyl alcohol was heated at reflux for 6 days. The solution wascooled to about 25° C. and the solvent was removed under reducedpressure to provide 4-hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)pyrazole-5-carboxamide.

M/e theory: 385; found: 385.

nmr (CDl₃): (125 Hz, m, 9H, 2',3', ##STR7##

EXAMPLE 8.

)

following the procedure set forth in Example 7, 4-hydroxy-3-β-D-(2',3',5'-tri-O-n-butyrylribofuranosyl)-N₁-n-butyrylpyrazole-5-carboxamide was converted to4-hydroxy-3-β-D-(2',3',5'-tri-O-n-butyrylribofuranosyl)pyrazole-5-carboxamide.

EXAMPLE 9 4-Hydroxy-3-β-D-ribofuranosylpyrazole-5-carboxamide 2',3'-acetonide

To a stirred suspension of 518 mg, of 4-hydroxy-3-β-D-ribofuranosylpyrazole-5-carboxamide and 15 cc. of acetone was addedin one portion 38 mg. of p-toluenesulfonic acid. The reaction mixturewas stirred for 16 hours at 25° C. Two cc. of 2,2-diethoxypropane wasadded to the reaction mixture in order to help drive the reaction tocompletion. The reaction mixture was stirred an additional 24 hours at25° C. To the stirred reaction mixture was added 1 cc. of concentratedammonium hydroxide solution, and stirring was continued for about 15minutes, after which time the solvent was removed under reducedpressure, affording a crude product. Further purification wasaccomplished by preparative thick layer chromatography of silica gelcoated glass plates, eluting with chloroform. The major component wasthe 2'3'-acetonide of 4-hydroxy-3-β-D-ribofuranosylpyrazole-5-carboxamide.

nmr (CDCl₃): (87 Hz and 100 Hz, 6H C(CH₃)₂)

EXAMPLE 104-Hydroxy-3-β-D-(5'-n-butyrylribofuranosyl)pyrazole-5-carboxamide.

A solution of 4-hydroxy-3-β-D-ribofuranosyl-N₁ -acetylpyrazole-5-carboxamide 2',3'-acetonide in 5 cc. of dry pyradine was stirred andcooled to 0° C. while 1 cc. of butyric anhydride was added to thereaction mixture during 2 minutes. The reaction mixture was stirred for6 hours at 0° C. The solvents were removed under reduced pressure,affording 4-hydroxy-3-β-D-(5'-O-n-butyrylribofuranosyl)-N₁-acetylpyrazole-5-carboxamide 2',3'-acetonide as a residue, which wasthen dissolved in 20 cc. of 50 percent aqueous methanol and heated atreflux for 10 hours. The solvents were removed under reduced pressureand the product was dissolved in 25 cc. 50 percent methanol-toluene. Thesolvents were again removed under reduced pressure, providing4-hydroxy-3-β -D-(5'-O-butyrylribofuranosyl)pyrazole-5-carboxamide.

EXAMPLE 11 4-Hydroxy-3-β-D-(5'-O-palmitoyribofuranosyl)pyrazole-5-carboxamide.

A solution of 319 mg. of 4-hydroxy-3-β-D-ribofuranosyl-N.sub. 1-acetylpyrazole-5-carboxamide 2',3'-acetonide was dissolved in 5 cc. ofdimethylacetamide containing 325 mg. of palmitoyl chloride. The reactionmixture was stirred at room temperature for 72 hours. The solvent wasremoved under reduced pressure, providing an oily residue which wasdissolved in 20 cc. of 50 percent aqueous methanol and stirred for 16hours at room temperature. The solvent was removed under reducedpressure, and the product was extracted into chloroform and washed withsaturated aqueous sodium bicarbonate solution and with water, and dried.Removal of the solvent under reduced pressure provided4-hydroxy-3-β-D-(5'-O-palmitoylribofuranosyl)pyrazole-5-carboxamide.

EXAMPLE 12 4-Hydroxy-3β-D-(2',3', 5'-tri-O-acetyribofuranosyl)-N₁-acetylpyrazole-5-N-acetylcarboxamide.

A solution of 1.0 g. of 4-hydroxy-3-β-D-ribofuranosyl-N.sub. 1-acetypyrazole-5-carboxamide in 10 cc. of acetic anhydride was stirredand cooled to 0° C. and 3 cc of triethylamine was added in one portionto the reaction mixture. The reaction mixture was allowed to warm to 25°C. and stirring was continued for 48 hours. Removal of the solvent underreduced pressure provided an oily residue which was redissolved in 25cc. of a 50 percent solution of benzene-dichloromethane. The solvent wasagain removed under reduced pressure. The residue was dissolved in 50cc. of chloroform and washed with water, 0.1 N hydrochloric acidsolution, saturated aqueous sodium bicarbonate solution, and dried.Removal of the solvent under reduced pressure provided a thick syrupwhich was then dissolved in 2 cc. of chloroform and applied to a columnpacked with 30 g. of silica gel (Woelm Grade 1). The column was firsteluted with 1 liter of hexane, followed by elution with 1 liter of 50percent hexane-benzene, then with 1 liter of 90 percent benzene-ethylacetate, 1 liter of 75 percent benzene-ethyl acetate, 1 liter of 50percent benzene-ethyl acetate, and finally with 1 liter of ethylacetate. The fractions eluted with 50 percent benzene-ethyl acetate werecombined and the solvent was removed under reduced pressure, providing800 mg. of 4-hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)-N.sub. 1-acetylpyrazole-5-N-acetylcarboxamide. ##STR8##

The ethyl acetate eluate fractions were combined and the solvent wasremoved therefrom under reduced pressure, affording 395 mg. of4-hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)pyrazole-5-N-acetylcarboxamide. ##STR9##

EXAMPLE 13 4-Hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)pyrazole-5-N-acetycarboxamide

Triethylamine was added to a solution of 4-hydroxy-3-β-D-ribofuranosyl-N₁ -acetylpyrazole-5-carboxamide in acetic anhydrideat 0° C. The reaction mixture was warmed to about 10° C. and stirred for1 hour. The reaction mixture was concentrated under reduced pressure toprovide an oily residue which was redissolved in abenzene-dichloromethane solution and again the solvent was removed. Theresulting residue was dissolved in a 20 percent methanol in watersolution and stirred for 5 hours. The solvents were completely removedunder reduced pressure to provide 4-hydroxy-3-β-D-(2',3',5'-tri-O-acetylribofuranosyl)pyrazole-5-N-acetylcarboxamide.

I claim:
 1. A process for preparing a pyrazofurin mono-N₁ -acylate of the formula ##STR10## wherein R₁ is C₁ -C₆ alkanoyl, comprising treating pyrazofurin with an acylating agent in an alcoholic solvent at a temperature of about 0° C. to about 30° C. for a period of time of about 30 to about 90 minutes. 